Delivery of a hammerhead ribozyme specifically down-regulates the production of fibrillin-1 by cultured dermal fibroblasts

The hammerhead ribozyme is a small catalytic RNA molecule. Potential hammerhead ribozymes that possess a catalytic domain and flanking sequence complementary to a target mRNA can cleave in trans at a putative cleavage site within the target molecule. We have investigated the potential of hammerhead ribozymes to down-regulate the product of the fibrillin-1 gene (FBN1). Fibrillin is a 347 kDa glycoprotein that is a major constituent of the elastin-associated microfibrils. Mutations in the FBN1 gene are responsible for Marfan syndrome (MFS), a common systemic disorder of the connective tissue. Many FBN1 mutations responsible for MFS appear to act in a dominant-negative fashion, raising the possibility that reduction of the amount of product from the mutant FBN1 allele might be a valid therapeutic approach for MFS. A trans-acting hammerhead ribozyme (FBN1-RZ1) targeted to the 5' end of the human FBN1 mRNA has been designed and synthesized, and shown to cleave its target efficiently in vitro. FBN1-RZ1 cleavage is magnesium dependent and efficient at both 37 and 50 degrees C. Delivery of the FBN1-RZ1 ribozyme into cultured dermal fibroblasts, by receptor- mediated endocytosis of a ribozyme-transferrin-polylysine complex, specifically reduces both cellular FBN1 mRNA and the deposition of fibrillin in the extracellular matrix. These results suggest that the use of hammerhead ribozymes is a valid approach to the study of fibrillin gene expression and possibly to the development of a therapeutic approach to MFS.      

Cloning and developmental expression analysis of the murine homolog of the spinocerebellar ataxia type 1 gene (Sca1)

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat which encodes glutamine in the novel protein ataxin-1. In order to characterize the developmental expression pattern of SCA1 and to identify putative functional domains in ataxin-1, the murine homolog (Sca1) was isolated. Cloning and characterization of the murine Sca1 gene revealed that the gene organization is similar to that of the human gene. The murine and human ataxin-1 are highly homologous but the CAG repeat is virtually absent in the mouse sequence suggesting that the polyglutamine stretch is not essential for the normal function of ataxin-1 in mice. Cellular and developmental expression of the murine homolog was examined using RNA in situ hybridization. During cerebellar development, there is a transient burst of Sca1 expression at postnatal day 14 when the murine cerebellar cortex becomes physiologically functional. There is also marked expression of Sca1 in mesenchymal cells of the intervertebral discs during development of the spinal column. These results suggest that the normal Sca1 gene, has a role at specific stages of both cerebellar and vertebral column development.      

Reliability of the acetabular teardrop as a landmark

Summary This study investigated the effect of tilt and observer reliability on radiographic measurements of the position of a prosthetic acetabular cup in seven dry bone pelves using the teardrop as a landmark. Coronal or sagittal tilt of more than five degrees was easily recognisable and there was effectively no observer variation in the measurements up to this limit. In addition, 90 out of 100 randomly selected antero-posterior pelvic radiographs from an outpatient department were not significantly rotated and 93 demonstrated a clearly defined teardrop. Measurements about the teardrop on routine radiographs are therefore sufficiently accurate to allow assessment of prosthetic position.

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La valeur du sourcil cotyloidien comme repère d'analyse radiologique

Résumé Cette étude, conduite sur 7 bassins secs, apprécie l'effet de l'inclinaison du bassin sur la qualité de l'analyse radiographique de la position d'une cupule prothétique de hanche en utilisant le sourcil cotyloïdien comme repère. Une inclinaison du bassin dans les plans coronal et sagittal est aisément détectable et il n'existe pas de variation d'analyse entre les différents observateurs en dessous de 5° d'inclinaison. De plus, sur 100 radiographies antéro-postérieures de bassin choisies au hasard dans les dossiers de consultation, 90 avaient été réalisées sans incidence particulièrement adaptée et l'on pouvait repérer facilement le sourcil sur 93% d'entre elles. Les mesures faites sur des radios de routine sont donc suffisamment précises pour permettre l'évaluation de la position d'une prothèse à partir du sourcil cotyloïdien.

     

Placental Thrombosis and Fetal Loss After Passive Transfer of Mouse Lupus Monoclonal or Human Polyclonal Anti-Cardiolipin Antibodies in Pregnant Naive BALB/c Mice

In the present study we evaluated the effect of passive transfer of a mouse monoclonal (CAM) or a human polyclonal anti-cardiolipin IgG on pregnancy outcome in BALB/c mice. The mice were immunized through the tail vein immediately after mating with 10 μg of monoclonal or polyclonal anti-cardiolipin antibodies. Two other groups of mice were given a mouse irrelevant monoclonal antibody or normal human polyclonal IgG respectively, at the same dose. In mice immunized with monoclonal or polyclonal anti-cardiolipin antibody we observed a significant increase in the number of fetal resorptions and a significant reduction of the mean weights of the embryos and the placentas. In mice immunized with CAM we also found a significant decrease in the number of healthy pups, while mice infused with human aCL antibody expressed a significant reduction in the fecundity rate. The histological examination showed widespread thrombosis and necrosis in the placentas derived from the mice immunized with the anti-cardiolipin antibodies. The model supports a possible direct pathogenetic effect of anti-phospholipid antibodies in recurrent fetal loss and points out that thrombotic events at placental level can be instrumental in the pathogenesis of the obstetric complications.     

Cloning and expression of the gene involved in Sanfilippo B syndrome (mucopolysaccharidosis III B)

Sanfilippo B syndrome is caused by a deficiency of alpha-N- acetylglucosaminidase, a lysosomal enzyme involved in the degradation of heparan sulphate. Accumulation of the substrate in lysosomes results in degeneration of the central nervous system with progressive dementia often combined with hyperactivity and aggressive behaviour. In order to clone the deficient gene, we purified the enzyme from human placenta and obtained amino acid sequence information. Alignment of one of the CNBr generated internal peptides to sequence from the database revealed the chromosomal location of the gene in the 5' upstream flanking region of the gene for 17-beta-hydroxysteroid-dehydrogenase at 17q21.1. The available DNA sequence was used to clone the cDNA coding for alpha-N- acetylglucosaminidase and analyse its gene structure. The gene is fully contained in the 5' upstream flanking region of the gene for 17-beta- hydroxysteroid-dehydrogenase and interrupted by five introns. The cDNA clone has a length of 2575 bp and encodes a protein of 743 amino acids. Chinese hamster ovary cells transfected with the cDNA construct show alpha-N-acetylglucosaminidase activity about 17-fold over background. This will allow correction studies with NAG deficient Sanfilippo B cell lines and facilitate the development of enzyme replacement therapy for these patients.      

Ceratobium mosaic potyvirus: another virus from orchids

Summary.  A potyvirus, which we call ceratobium mosaic virus, has been detected in about one third of more than 100 plants representing c. 33 orchid genera in two collections in Australia. It was detected using RT-PCR with redundant primers that are Potyviridae-specific and have additional sequences corresponding to either the SP6 or T7 bacteriophage promoters at their 5′-termini. Thus the nucleotide sequence of the resulting PCR fragments, consisting of about 1.7 kb of the 3′ portion of the viral genome, could be determined directly. Viral sequences obtained from five infected orchids indicate that they contained different isolates of a single potyvirus species most closely related to the bean common mosaic group of potyviruses, but clearly distinct from all whose virion protein genes have been reported to the international gene sequence databases. Accepted December 18, 1997 Received September 9, 1997     

Identification of MEN1 gene mutations in sporadic carcinoid tumors of the lung

Lung carcinoids occur sporadically and rarely in association with multiple endocrine neoplasia type 1 (MEN1). There are no well defined genetic abnormalities known to occur in these tumors. We studied 11 sporadic lung carcinoids for loss of heterozygosity (LOH) at the locus of the MEN1 gene on chromosome 11q13, and for mutations of the MEN1 gene using dideoxy fingerprinting. Additionally, a lung carcinoid from a MEN1 patient was studied. In four of 11 (36%) sporadic tumors, both copies of the MEN1 gene were inactivated. All four tumors showed the presence of a MEN1 gene mutation and loss of the other allele. Observed mutations included a 1 bp insertion, a 1 bp deletion, a 13 bp deletion and a single nucleotide substitution affecting a donor splice site. Each mutation predicts truncation or potentially complete loss of menin. The remaining seven tumors showed neither the presence of a MEN1 gene mutation nor 11q13 LOH. The tumor from the MEN1 patient showed LOH at chromosome 11q13 and a complex germline MEN1 gene mutation. The data implicate the MEN1 gene in the pathogenesis of sporadic lung carcinoids, representing the first defined genetic alteration in these tumors.      

Neutralizing antibody spectrum determines the antigenic profiles of emerging mutants of visna virus.

The visna viruses are antigenically related nononcogenic retroviruses of sheep. The original strain was isolated from the brain of a paralyzed sheep in Iceland during the 1940s. The prototype strains has been passed serially in sheep and has undergone progressive antigenic change. Previous reports have shown that such antigenic changes in visna virus can be reproduced in infected cell cultures treated with neutralizing antibody. We now show that the antigenic profiles of the emerging mutants directly reflect the nature of the selecting antibody. Mutants with minor antigenic changes were selected by "early" sera which had a limited neutralization range. Mutants with greater antigenic changes were selected by "late" sera with a wide neutralization range. Mutants selected by early sera emerged rapidly and consistently in cultures, and these were antigenically very similar to one another. Mutants rarely emerged in cultures treated with late sera, but these viruses showed major antigenic changes. The data suggest that the evolution of antigenic mutants of visna virus progresses by a series of minor mutations which accumulate under the selective pressure of antibody.     

微乳液反应法制备磺胺嘧啶银均匀微晶及其最佳处方评价

目的：应用微乳液反应法制备磺胺嘧啶银均匀微晶,均匀制得的微晶的粒径大小约为2～4um,均匀微晶的结晶性好,纯度高。用均匀设计方法优化条件,制备的均匀的微晶平均粒径大小为2.09um,实验结果达到预测结果要求。结论：用微乳液反应法能获得磺胺嘧啶银均匀微晶。     

Role of Tris-CaEDTA as an adjuvant with nebulised tobramycin in cystic fibrosis patients with Pseudomonas aeruginosa lung infections: A randomised controlled trial

BackgroundWe tested if disrupting iron utilisation by P. aeruginosa by adding the Tris-buffered chelating agent CaEDTA to nebulised tobramycin would enhance bacterial clearance and improve lung function in CF patients.MethodsIn this double-blind, randomised controlled trial, 26 episodes (25 patients) with P. aeruginosa infection admitted to two CF centres for treatment of an acute pulmonary exacerbation were randomly assigned to receive either 75 mg CaEDTA in Tris-buffered saline or placebo (Tris-buffered saline) nebulised in combination with 250 mg tobramycin twice daily for six weeks followed with four week safety follow-up. Primary endpoints were safety, tolerability, and bacterial density of P. aeruginosa. A secondary endpoint was lung function.ResultsThe study drug was well tolerated with adverse events comparable in both groups. The mean (SD) reduction in sputum P. aeruginosa count (log₁₀ CFU/g) in the CaEDTA vs placebo group was 2·05 (2·57) vs 0·82 (2·71) at two weeks relative to admission (p = 0·39). The mean improvement in ppFEV₁ was 16 vs 5 (p = 0·16); 11 vs 2 (p = 0·28); and 6 vs 2 percentage points (p = 0·47) at two, six, and ten weeks in CaEDTA and placebo groups, respectively.ConclusionsIn this pilot study in CF patients, an increase in the reduction of sputum density of P. aeruginosa and an increase in ppFEV₁ was observed in the group of patients who received Tris-CaEDTA added to inhaled tobramycin compared to the group who received inhaled tobramycin alone, although these differences were not statistically significant. The treatment was also shown to be safe.